This project aims to develop fluorescent monoclonal antibodies as biomarkers for living, human cholinergic neurons and their precursors, in vitro. The most important use of these antibodies will be for identifying human stem cells that are undergoing cholinergic differentiation. Success of this project depends on antibodies that bind to cell surface markers that are preferentially expressed by cholinergic neurons (and their progenitors). Currently, the high affinity choline transporter is the only cell surface antigen that is recognized as being universally expressed by cholinergic neurons. Thus, mice will be immunized with peptide immunogens that correspond to specific extracellular regions of the human, high affinity choline transporter. Hybridoma supernatants from these animals will be screened for their ability to detect the human high affinity choline transporter that is expressed in frog oocytes, HEK-293 cells and in fixed human brain sections. Antibodies that satisfy empirical criteria of specificity and sensitivity will then be fluorescently labeled and evaluated for their utility in detecting the cholinergic differentiation of human stem cells in culture. Antibodies that are viable in this context will be distributed to the stem cell community as tools to improve protocols for obtaining stem cell-derived cholinergic neurons. Such cholinergic neuron precursors are expected to be useful for cell replacement therapies in disorders that involve a loss of cholinergic neurons (like amyotrophic lateral sclerosis and Alzheimer's disease). Thus, this project should serve to catalyze progress toward stem cell-based therapies for disorders that affect cholinergic neurons. PUBLIC HEALTH RELEVANCE: Alzheimer's disease and amyotrophic lateral sclerosis are devastating neurodegenerative disorders that involve a loss of cholinergic neurons. The antibody biomarkers developed by this project will facilitate the identification and characterization of living, human cholinergic neurons (and possibly, their precursors) that are derived from stem cells. The widespread dissemination of these biomarkers to the research community should significantly advance efforts geared to the use of cell-replacement therapies for these diseases.